Large-scale production of lentiviral vectors using multilayer cell factories
Main Article Content
Keywords
calcium phosphate, cell factories, lentiviral vector, Mustang Q, ultracentrifugation
Abstract
Lentiviral-mediated gene therapy has been proposed for the treatment of a range of diseases, and due to its genome integration properties, it offers the potential for long-lasting benefit from a once-off treatment. Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNCâ„¢ EasyFillâ„¢ Cell Factoryâ„¢ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 108–109 TU/ml, with reduced manual handling compared to single monolayer flask methods.
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References
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2. Biffi A, Montini E, Lorioli L, Cesani M, Fumagalli F, Plati T, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science. 2013;341(6148):1233158.
3. Hacein-Bey Abina S, Gaspar HB, Blondeau J, Caccavelli L, Charrier S, Buckland K, et al. Outcomes following gene therapy in patients with severe Wiskott-Aldrich syndrome. JAMA. 2015;313(15):1550-63.
4. Aiuti A, Biasco L, Scaramuzza S, Ferrua F, Cicalese MP, Baricordi C, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science. 2013;341(6148):1233151.
5. Palfi S, Gurruchaga JM, Ralph GS, Lepetit H, Lavisse S, Buttery PC, et al. Long-term safety and tolerability of ProSavin, a lentiviral vector-based gene therapy for Parkinson's disease: a dose escalation, open-label, phase 1/2 trial. Lancet. 2014;383(9923):1138-46.
6. Malik P. Gene Therapy for Hemoglobinopathies: Tremendous Successes and Remaining Caveats. Mol Ther. 2016;24(4):668-70.
7. Negre O, Eggimann AV, Beuzard Y, Ribeil JA, Bourget P, Borwornpinyo S, et al. Gene Therapy of the beta-Hemoglobinopathies by Lentiviral Transfer of the beta(A(T87Q))-Globin Gene. Hum Gene Ther. 2016;27(2):148-65.
8. Fuller M, Anson DS. Helper plasmids for production of HIV-1-derived vectors. Hum Gene Ther. 2001;12(17):2081-93.
9. McIntyre C, Derrick-Roberts AL, Byers S, Anson DS. Correction of murine mucopolysaccharidosis type IIIA central nervous system pathology by intracerebroventricular lentiviral-mediated gene delivery. J Gene Med. 2014;16(11-12):374-87.
10. Derrick-Roberts AL, Pyragius CE, Kaidonis XM, Jackson MR, Anson DS, Byers S. Lentiviral-mediated gene therapy results in sustained expression of beta-glucuronidase for up to 12 months in the gus(mps/mps) and up to 18 months in the gus(tm(L175F)Sly) mouse models of mucopolysaccharidosis type VII. Hum Gene Ther. 2014;25(9):798-810.
11. Cmielewski P, Donnelley M, Parsons DW. Long-term therapeutic and reporter gene expression in lentiviral vector treated cystic fibrosis mice. J Gene Med. 2014;16(9-10):291-9.
12. Wong ES, McIntyre C, Peters HL, Ranieri E, Anson DS, Fletcher JM. Correction of methylmalonic aciduria in vivo using a codon-optimized lentiviral vector. Hum Gene Ther. 2014;25(6):529-38.
13. Merten OW, Hebben M, Bovolenta C. Production of lentiviral vectors. Mol Ther Methods Clin Dev. 2016;3:16017.
14. Merten OW. Advances in cell culture: anchorage dependence. Philos Trans R Soc Lond B Biol Sci. 2015;370(1661):20140040.
15. Segura MM, Mangion M, Gaillet B, Garnier A. New developments in lentiviral vector design, production and purification. Expert Opin Biol Ther. 2013;13(7):987-1011.
16. Segura MM, Garnier A, Durocher Y, Coelho H, Kamen A. Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification. Biotechnol Bioeng. 2007;98(4):789-99.
17. Ansorge S, Lanthier S, Transfiguracion J, Durocher Y, Henry O, Kamen A. Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures. J Gene Med. 2009;11(10):868-76.
18. McKay T, Patel M, Pickles RJ, Johnson LG, Olsen JC. Influenza M2 envelope protein augments avian influenza hemagglutinin pseudotyping of lentiviral vectors. Gene Ther. 2006;13(8):715-24.
19. Matsui H, Hegadorn C, Ozelo M, Burnett E, Tuttle A, Labelle A, et al. A microRNA-regulated and GP64-pseudotyped lentiviral vector mediates stable expression of FVIII in a murine model of Hemophilia A. Mol Ther. 2011;19(4):723-30.
20. Gutierrez-Granados S, Cervera L, Segura Mde L, Wolfel J, Godia F. Optimized production of HIV-1 virus-like particles by transient transfection in CAP-T cells. Appl Microbiol Biotechnol. 2016;100(9):3935-47.
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Published
Apr 10, 2018
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Rout-Pitt N, McCarron A, McIntyre C, Parsons D, Donnelley M. Large-scale production of lentiviral vectors using multilayer cell factories. J Biol Methods [Internet]. 2018 Apr. 10 [cited 2024 Apr. 19];5(2):e90. Available from: https://jbmethods.org/index.php/jbm/article/view/236
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